PUBLICATION

Effect of cryopreservation on mitochondrial DNA of zebrafish (Danio rerio) blastomere cells

Authors
Kopeika, J., Zhang, T., Rawson, D.M., and Elgar, G.
ID
ZDB-PUB-050202-5
Date
2005
Source
Mutation research   570(1): 49-61 (Journal)
Registered Authors
Elgar, Greg, Rawson, David M., Zhang, Tiantian
Keywords
Cryopreservation; Blastomeres; Mitochondrial DNA; Zebrafish
MeSH Terms
  • Animals
  • Base Sequence
  • Blastomeres*
  • Cryopreservation*
  • DNA Mutational Analysis
  • DNA, Mitochondrial*
  • Mitochondria/genetics*
  • Molecular Sequence Data
  • Mutation
  • Sequence Alignment
  • Zebrafish
PubMed
15680402 Full text @ Mutat. Res.
Abstract
Cryopreservation has been extensively used in human reproductive medicine, aquaculture and conservation programmes for endangered species. However, despite the growing successes of cryopreservation, post-thaw recovery of reproductive and embryonic cells very often remains poor. Many studies have been devoted to the mechanisms of cryodamage. It is known that cryopreservation causes extensive damage to membranes; reduce the metabolic activity of cells; and disturbs the mitochondrial bioenergetical processes of cells. But few investigations on the genetic stability of cells during cryopreservation have been performed, and the role of any genetic impact cryopreservation needs to be determined. Some indirect data in the literature suggests that progress in this field might come from investigating freezing damage to mitochondrial DNA (mtDNA), nuclear DNA and other genome-related structures. In this study, zebrafish (Danio rerio) blastomeres were treated in three different ways: control suspension of blastomere cells in phosphate buffered saline; equilibration of blastomeres with 2M dimethyl sulfoxide (Me(2)SO) for 1h at room temperature and cryopreservation using Me(2)SO as a cryoprotectant. Mitochondrial DNA was analysed in fresh cells and after the different treatments. Two different loci of mtDNA were amplified with the help of PCR and sequenced. The sequences were analysed and nuclear base substitutions were counted for both control and treated samples. The results showed that cryopreservation significantly increased the frequency of mutations (0.78+/-0.27% in comparison to 0.16+/-0.25% of control), whilst 2M Me(2)SO treatment did not bring a significant increase in frequency of mutations (0.24+/-0.28%). The distributions of the mutation locations were analysed. More investigations are needed to determine whether optimisation of cryopreservation protocol is possible to reduce these adverse effects; whether such mutations interfere with overall function of the cells; whether similar changes also occur in the nuclear DNA and whether such mutations happen in other species. Meanwhile, it is important to be cautious in making judgements of the effect of cryopreservation technique in assisted reproduction. This is the first report on the effect of cryopreservation on mtDNA.
Genes / Markers
Figures
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping